Methyl salicylate-based attractants for vectors of citrus greening disease

ABSTRACT

The present invention is directed to an insect attractant having an amount of methyl salicylate effective to attract a plurality of vectors of  citrus  greening disease and an agriculturally acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Application 61/601,617filed Feb. 22, 2012, to which priority is claimed and whose teachingsare incorporated herein.

FIELD OF THE INVENTION

The present invention relates to insect attractants, and moreparticularly to attractants for vectors of Huanglongbing (HLB) (citrusgreening disease), methods for monitoring infestation of citrus plants,and to methods for diverting vectors of HLB away from a predeterminedcitrus growth.

BACKGROUND OF THE INVENTION

HLB is the most devastating disease of citrus worldwide. HLB affectsplant phloem, causing yellow shoots, mottling, chlorosis, twig die backthat result in rapid tree decline and may ultimately cause tree death.Fruit on diseased trees are misshaped, reduced in size, and are sour intaste (Capoor, 1963; Halbert & Manjunath, 2004; Bové, 2006; Dagulo,2010). The disease is associated with either of three species ofphloem-limited, noncultured, Gram-negative bacteria, CandidatusLiberibacter asiaticus (Las), Candidatus Liberibacter africanus (Laf) orCandidatus Liberibacter americanus (Lam) (Bove, 2006; Gottwald, 2010).HLB is primarily vectored by Asian citrus psyllid, Diaphorina citriKuwayama (Hemiptera, Psyllidae); however, African citrus psyllid, Triosaerytreae, is also known to transmit the bacteria in Africa. Both psyllidspecies can transmit any of the three bacterial species (Gotwald, 2010).In North America, the disease is thought to be mainly caused by Ca. L.asiaticus and vectored by D. citri (Bové, 2006; Gottwald et al., 2007;Gottwald, 2010).

Current management for D. citri primarily relies on broad spectruminsecticide applications (Roger, 2008) because of a lack of knownresistant cultivars (Halbert & Manjunath 2004), effective biologicalcontrol agents (Qureshi & Stansly 2007) or cultural control options(Childers & Rogers 2005; Powell et al., 2007). However, insecticide usehas negatively affected populations of natural enemies and has led todevelopment of insecticide resistance (Tiwari et al., 2011). Therefore,identification of attractants may allow development of usefulalternatives or supplements to conventional insecticides. Recentinvestigations indicate that males of some psyllid species use volatilechemicals to locate mates (Soroker et al., 2004; Wenninger et al., 2008;Guedot et al., 2009). Behavioral evidence for a female-produced volatilesex attractant was reported in pear psylla C. bidens (Soroker et al.,2004). Post-diapausing female pear psylla, Cacopsylla pyricola (Förster)produces a cuticular hydrocarbon, 13-methylheptacosane, that attractsopposite sex conspecifics (Guédot et al., 2009). Behavioral evidence ofa female derived olfactory attractant for male D. citri was also shownin laboratory olfactometer assays (Wenninger et al., 2008). Furthermore,ultrastructure of olfactory sensilla on D. citri antennae suggestschemosensory functions (Onagbola et al., 2008).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the response of D. citri to odors emitted from Las-infectedversus non-infected citrus in a laboratory olfactometer. Bars labeled byan asterisk are significantly different (χ2 test, p<0.05). n=totalnumber of psyllids that responded.

FIG. 2 shows the settling preference of combined non-infected andLas-infected D. citri on Las-infected versus non-infected citrus plants.Panel (A) shows response under light conditions and panel (B) showsresponse under dark conditions. Bars with the same letter are notsignificantly different (Tukey's HSD test, p<0.05).

FIG. 3 shows the movement of previously settled D. citri fromLas-infected to non-infected citrus plants. Panel (A) shows response ofnon-infected psyllids and panel (B) shows response of Las-infectedpsyllids. Bars labeled with different letters are significantlydifferent from one another (Tukey's HSD test, p<0.05).

FIG. 4 shows the feeding efficiency of D. citri on Las-infected versusnon-infected citrus leaves as measured by honeydew excretion. Barslabeled with different letters are significantly different from oneanother (Tukey's HSD test, p<0.05).

FIG. 5 shows chromatograms displaying volatile differences betweenLas-infected and non-infected plants. Release of methyl salicylate issignificantly greater from plants infected with Las, while release ofD-limonene and methyl anthranilate is significantly greater fromnon-infected plants.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified specific mechanisms through whicha bacterial plant pathogen induces plant responses that modify behaviorof its insect vector. For example, the present inventors found thatCandidatus Liberibacter asiaticus, a fastidious, phloem-limitedbacterium responsible for causing citrus greening disease, inducedrelease of a specific volatile chemical, methyl salicylate, whichincreased attractiveness of infected plants to its insect vector,Diaphorina citri, and caused vectors to initially prefer infectedplants. The insect vectors subsequently dispersed to non-infected plantsas their preferred location of prolonged settling because of likelysub-optimal nutritional content of infected plants. The duration ofinitial feeding on infected plants was sufficiently long for the vectorsto acquire the pathogen before they dispersed to non-infected plants,suggesting that the bacterial pathogen manipulates behavior of itsinsect vector to promote its own proliferation. The behavior of psyllidsin response to infected versus non-infected plants was not influenced bywhether or not they were carriers of the pathogen and was similar underboth light and dark conditions. Feeding on citrus by D. citri adultsalso induced the release of methyl salicylate, suggesting that methylsalicylate is a cue revealing location of conspecifics on host plants.The present inventors have thus identified specific attractants, e.g.,methyl salicylate, for D. citri that may have practical applications,such as for use in monitoring the presence of D. citri in specificcitrus growth areas prior to insecticide application. In addition, theidentified attractants may be provided as a component of an attractantcomposition to draw the harmful D. citri away from the target crop.

As used herein, the terms “attract” or “attractant” mean that, as aresult of the presence of an attractant as described herein, a greaternumber of vectors of citrus greening disease are present in a definedarea than would be present without the attractant.

As used herein, the term “methyl salicylate” (lauric acid) refers tomethyl salicylate and derivatives or analogs thereof.

As used herein, by “effective amount,” “amount effective,” or the like,it is meant an amount effective at dosages and for periods of timenecessary to achieve the desired result, e.g., attracting a plurality ofvectors of citrus greening disease.

By “repel” or “repelling” as used herein, it is meant that there areless vectors of citrus greening disease in a desired area than therewould have been if the attractant were not present. Repel or repellingalso includes the prevention of an infestation by an action in a desiredarea where there are no pests present, but at least one pest would bepresent if not for the action taken.

By “killing” as used herein, it is meant that a composition kills thevectors of citrus greening disease (pests) and/or inhibits or reducesthe growth of the vectors. The growth of a pest has been inhibited ifthere has been a relative reduction in the number of pests in a desiredarea. The growth of a pest may also be said to have been inhibited ifthe normal growth pattern of the pest has been modified so as to have anegative effect on the individual pest. The number of pests has beenreduced by an action if there are fewer pests in a desired area thanthere would have been without the action.

In accordance with an aspect of the present invention, there is providedan insect attractant comprising an amount of methyl salicylate effectiveto attract a plurality of vectors of citrus greening disease. The methylsalicylate may be provided from any suitable source or via any synthesismethod known in the art. In one embodiment, the methyl salicylate may beprovided from any suitable commercial source, such as Sigma Aldrich. Theamount of methyl salicylate in the attractant composition may be from0.001 μg to 10,000 μg, and in one embodiment, and in a preferredembodiment, is from 0.001 to 100 μg.

In one embodiment, the attractant composition may comprise furthercomponents, which may be useful as attractants for vectors of citrusgreening disease. For example, in one embodiment, the attractant mayfurther comprise a compound selected from the group consisting ofβ-ocimene, D-limonene, methyl anthranilate, and combinations thereof.β-ocimene and D-limonene are predominant citrus volatiles released bycitrus flush, foliage, and fruit [38,63-65]. Methyl anthranilate wasfound to be released in significantly greater quantities relative tonon-infected plants and may have attractant properties.

The attractants described herein may be formulated into compositionsaccording to known methods for formulating agricultural/horticulturalcompounds. In one embodiment, the methyl salicylate (and any otheractive compounds capable of attracting vectors of citrus greeningdisease) may be mixed with an appropriate agriculturally acceptablecarrier, and if required, an auxiliary at a proper proportion. Theresultant mixture may be subjected to dissolution, separation,suspension, mixing, impregnation, adsorption or adhesion and can beformulated into any desired forms for practical use, such as solubleconcentrates, emulsifiable concentrates, wettable powders, water solublepowders, water dispersible granules, water soluble granules, suspensionconcentrates, concentrated emulsions, suspoemulsions, microemulsions,dustable powders, granules, tablets and emulsifiable gels. By“agriculturally acceptable carrier,” it is meant an agent that does nothave a substantial detrimental effect on the activity of the activeingredients described herein.

Suitable solid agriculturally acceptable carriers include, but are notlimited to, soybean flour, grain flour, wood flour, bark flour, sawingflour, tobacco stalk flour, walnut shell flour, bran, cellulose powder,a residue after plant extraction, a synthetic polymer such as asynthetic resin powder, clay (e.g., kaoline, bentonite, or acid whiteclay), talc (e.g., talc or pyrophyllite), silica (e.g., diatomite,silica powder, mica, activated carbon, sulfur powder, pumice, calcineddiatomite, brick powder, fly ash, sand, inorganic mineral powders suchas calcium carbonate and calcium phosphate, chemical fertilizers such asammonium sulfate, ammonium phosphate, ammonoium nitrate, urea, andammonium chloride, and compost.

Suitable liquid carriers may be one having a solvent ability or amaterial having no solvent ability for the components, including methylsalicylate, but having an ability to assist in the dispersion of theactive ingredient compound, e.g., methyl salicylate. Exemplary liquidcarriers include but are not limited to, alcohols (e.g., methanol,ethanol, isopropanol, butanol, and ethylene glycol); ketones (e.g.,acetone, methylethyl ketone, methyl isobutyl ketone, diisobutyl ketone,and cyclohexanone); ethers (e.g., diethyl ether, dioxane, cellosolve,diisopropyl ether, and tetrahydrofuran); aliphatic hydrocarbons (e.g.,kerosine and mineral oil); aromatic hydrocarbons (e.g. benzene, toluene,xylene, solvent naphtha, and alkylnaphthalene); halogenated hydrocarbons(e.g., dichloromethane, chloroform, carbon tetrachloride, andchlorobenzene); esters (e.g., ethyl acetate, butyl acetate, ethylpropionate, diisobutyl phthalate, dibutyl phthalate, and dioctylphthalate); amides (e.g., dimethylformamide, diethylformamide, anddimethylacetamide); and nitriles (e.g., acetonitrile). In one particularembodiment, the agriculturally acceptable carrier comprises anagriculturally acceptable carrier oil, including but not limited to,mineral oil or a vegetable oil such as canola oil, sunflower oil,cottonseed oil, palm oil, soybean oil, and the like. In one furtherparticular embodiment, mineral oil is provided as the agriculturallyacceptable carrier.

When the insect attractant is to be in the form of an aerosol, apropellant may be added such as propane, butane, isobutane, dimethylether, carbon dioxide, nitrous oxide, nitrogen, and combinationsthereof. Further, it is understood that the compositions of the presentinvention may additionally include any suitable surfactant, penetratingagent, spreading agent, thickener, anti-freezing agent, binder,anti-caking agent, disintegrating agent, anti-foaming agent,preservative, stabilizer, and the like as is desired.

In accordance with another aspect of the present invention, there isprovided a method for inhibiting infestation of a citrus plant byvectors of citrus greening disease. The vectors may be, for example, theAsian citrus psyllid, Diaphorina citri (D. citri) or the African citruspsyllid, Triosa erytrae. These vectors carry at least one three speciesof phloem-limited, noncultures, Gram-negative bacteria, which may be oneof Candidatus Libreribacter asiaticus (Las), Candidatus Libreribacterafricanus (Laf), or Candidatus Libreribacter americanus (Lam). Las andLam are transmitted by D. citri; whereas, Laf is transmitted by Triozaerytreae. In one embodiment, the method comprises identifying a targetarea of desired citrus plant growth and applying an amount of an insectattractant comprising methyl salicylate to an area outside of the targetarea effective to attract the vectors of citrus greening disease awayfrom the target area.

The target area may be any sized area in which is desired to be free orsubstantially free of vectors of citrus greening disease. Typically,however, the target area is of a size with which the attractantcompositions described herein are sufficiently able to pull or attractvectors of citrus greening disease away from the target area to analternate area. The area outside of the target area is one where thevectors of citrus greening disease will not cause damage to citrusplants. The outside area may be barren, for example, or may containvegetation (plants, trees, or other) that are not affected by thevectors citrus greening area. In another embodiment, the outside areaincludes artificial vegetation. Typically, the location or locations ofapplication of the insect attractant lie a sufficient distance from thetarget area such that there is little possibility that the attractedvectors of citrus greening disease are drawn to the target area. Forexample, in one embodiment, the area outside of the target area is atleast 0.0001 m, 0.1 m, 0.5 m, 1 m, 2 m, 10 m, 50 m, 100 m, or 1000 mfrom a point on a perimeter of the target area. It is appreciated,however, that the attractant must, however, be sufficiently close to thetarget area for the vectors of citrus greening disease to sense theattractant and respond thereto.

It is contemplated that the application of the attractant to drawvectors of citrus greening disease may be done in conjunction with otherdisease management strategies (citrus greening treatment) known in theart for the repelling or killing vectors of citrus greening disease.Other suitable citrus disease management strategies, but are not limitedto, insecticide applications (Roger, 2008), biological control agents(Qureshi & Stansly 2007), and/or cultural control options (Childers &Rogers 2005; Powell et al., 2007). Exemplary compositions and methodsfor treating or preventing citrus greening disease are disclosed, forexample, in U.S. Published Patent Application No. 20110021463 and20100074972.

The attractant may be applied to the outside areas by any suitablemethod known in the art, such as by spray application, dusting or thelike. The application may be done with such quantities and durations tobring about the desired effect. In one embodiment, the application isdone by controlled release of the insect attractant through a metereddevice. In another embodiment where the attractant is at least partiallyvolatile, the attractant may be distributed from a container having aheating element to volatilize the sample, if necessary.

The citrus plants to be aided by the attractants described herein may beany tree or plant that is infected or may be infected with one or morevectors of citrus greening disease. The methods, compositions, andarticles of manufacture described herein are suitable for use on anytree or plant that is infected or may be infected with citrus greeningdisease. Exemplary plants include, by way of example only, any cultivarfrom the genus Citrus, including but not limited to Citrus sinensis,lemon (C. limon), lime (C. latifolia) grapefruit (C. paradise), sourorange (C. aurantium), and mandarin (C. reticulata).

In an additional embodiment, provided is a method that involves maskingsignals sent out to insect vectors as a result of the release of methylsalicylate from a disease tree. According to this embodiment, methylsalicylate is administered to a plurality of trees amongst which aninfected tree may exist. Since methyl salicylate is released as a resultof infection, and this serves as a beacon to attract insect vectors tocontribute spread of pathogen to non-infected trees, intentionallyapplying methyl salicylate across all of the trees will confuse theinsect vectors and diminish their ability to find and discern a treethat is infected from a tree that is not infected. This method can beapplied across a grove of trees to help ameliorate the spread ofpathogen because it significantly decreases the ability of insectvectors to find infected trees and transfer the infection to othertrees.

In a specific embodiment related to the preceding, methyl salicylate isadministered to a plurality of trees at an amount effect to mask signalsfrom an infected tree. More specifically, the effective may be at anamount that prevents the ability of an insect vector to discern betweenan infected tree and a non-infected tree. In a particular embodiment,methyl salicylate is administered at a concentration and amounteffective to attract insect vectors responsible for spread of citrusgreening.

Similar to that described above, the attractant may be applied to thetarget plants by any suitable method known in the art, such as by sprayapplication, dusting or the like. A slow release matrix, such as waxmatrix or microcapsules, may be used. The application may be done withsuch quantities and durations to bring about the desired effect. In oneembodiment, the application is done by controlled release of the insectattractant through a metered device. Examples of metered devices includebut are not limited to, spray bottles that are programmed to sprayintermittently, or devices that effuse the attractant over a period oftime. In another embodiment where the attractant is at least partiallyvolatile, the attractant may be distributed from a container having aheating element to volatilize the sample, if necessary. Thus, applying“to the trees” is to be interpreted as being applied directly on thetrees, or in the air around the trees.

The air around a tree would be air in contact with a tree surface or airfrom contact up to 1-10 feet away.

In an alternative embodiment, methyl salicylate is administered at aconcentration and amount effective to repel insect vectors. This wouldtypically be at amounts relatively higher than that effective to attractinsect vectors. Those skilled in the art will appreciate that theseconcentrations and amounts can be achieved in light of the teachingsherein.

In yet another aspect of the present invention, there is provided anarticle of manufacture for dispensing the attractant compositionsdescribed herein. The article of manufacture comprises an amount ofmethyl salicylate effective to attract a plurality of vectors of citrusgreening disease. Optionally, the article of manufacture furthercomprises an agriculturally acceptable carrier. Further, the article ofmanufacture comprises a container comprising the amount of methylsalicylate and the agriculturally acceptable carrier, if present, andmeans for applying the amount of methyl salicylate and theagriculturally acceptable carrier from the container to a predeterminedarea. The means for applying the methyl salicylate may comprise anysuitable spray applicator as is known in the art, for example.

In accordance with yet another aspect of the present invention, there isprovided a method for preventing or treating infestation of a citrusplant with vectors of citrus greening disease. The method comprisesdetecting an amount of methyl salicylate associated with the citrusplant. In one embodiment, the amount may be detected by capturing a headspace about the subject citrus plant(s) or growth. Thereafter, theamount of methyl salicylate present may be determined by knownchromatographic methods, including gas chromatography. The methodfurther requires that, upon detection of methyl salicylate, a citrusgreening disease treatment as described above may be applied to thecitrus plant in an amount effective to repel or kill the vectors ofcitrus greening disease by any suitable method as set forth above

The present inventors have also found that there were marked differencesbetween infected and non-infected plants with respect to nutrientcontent (Table 5 in the Example section). Las-infected plants weredeficient in N, P, Mg, Zn, and Fe as compared with non-infected plants(Table 5). However, Las-infected plants had higher K and B contentscompared with non-infected plants (Table 5). There were no differencesbetween Las-infected and non-infected plants for Ca, S, Si, Mn, Na, Mo,AL, and Cl. Accordingly, in accordance with another aspect of thepresent invention, there is provided a method for preventing or treatinginfestation of a citrus plant with vectors of citrus greening diseasethat detects the increase/decrease of such minerals in a citrus plant.Upon such detection, a citrus greening disease treatment may be appliedto the citrus plant in an amount effective to repel or kill the vectorsof citrus greening disease by any suitable method as set forth above.

The following examples are intended for the purpose of illustration ofthe present invention. However, the scope of the present inventionshould be defined as the claims appended hereto, and the followingexamples should not be construed as in any way limiting the scope of thepresent invention.

EXAMPLE 1 Materials and Methods Maintenance of Insect, Pathogen and HostPlants

Non-infected adult D. citri used in behavioral bioassays were obtainedfrom a laboratory culture at the University of Florida, Citrus Researchand Education Center (Lake Alfred, USA). The culture was established in2000 from field populations in Polk Co., FL, USA (28.0′N, 81.9′W) priorto the discovery of HLB in FL. The culture was maintained withoutexposure to insecticides on sour orange (Citrus aurantium L.) and‘Hamlin’ orange [C. sinensis (L.) Osb.]. Monthly testing of randomlysampled D. citri nymphs, adults, and plants by qPCR was conducted toconfirm that psyllids and plants in this culture are free of Las.

Las-infected D. citri were obtained from Las-infected C. aurantium andC. sinensis plants maintained in a secure quarantine facility at theUniversity of Florida, Citrus Research and Education Center. Routinesampling indicated that about 70% of D. citri obtained from this colonywere positive for Las when tested in qPCR assays. Both colonies weremaintained at 27±1° C., 63±2% RH, and under a L14:D10 hour photoperiod.Non-infected and Las-infected D. citri cultures were maintained indouble-screened, 3.7×4.6 m secure enclosures located in separatedbuildings and with minimal risk of cross contamination.

Las infection in host plants was maintained by graft-inoculation ofnon-infected C. sinensis with Las-infected key lime (Citrusaurantifolia) budwood collected from citrus groves in Immokalee, Fla.,USA. Grafted plants were tested for Las infection by qPCR four monthsafter grafting. Plants that tested positive for Las were used inexperiments and for maintenance of Las cultures. Cultures ofLas-infected plants were maintained through graft-inoculations becauseof low transmission efficiency of D. citri adults [17]. Because Las isnot seed transmissible [95], Las-free host plants used in experimentswere cultivated from C. sinensis seed or obtained as potted seedlingsfrom an HLB-free commercial nursery to minimize the risk of undetectablelatent infection of Las in grafted plants. The nursery-obtained plantswere confirmed negative for Las infection by qPCR. All infected plantsused for experiments exhibited minor or no symptoms, ranging from 0 to 1on a graded symptom scale of 1 to 10. Non-infected and Las-infectedplants were maintained in separate secure enclosures with minimal riskof cross contamination as described above.

Detection of Las in Insect and Plant Samples

Dual-labeled probes were used to detect Las in D. citri and citrusplants using an ABI 7500 system (Applied Biosystems, Foster City,Calif.) in a multiplex TaqMan quantitative real-time polymerase chainreaction (qPCR) assay described in [17,96]. DNA from insect and plantsamples was isolated using the DNeasy blood and tissue or DNeasy plantkits (Qiagen Inc, Valencia, Calif.), respectively. Las-specific 16S rDNAfrom psyllid and plant extracts was amplified using probe-primer setstargeting internal control sequences specific to D. citri [insectwingless] or plant [cytochrome oxidase] gene regions [17,96-97].

DNA amplifications were conducted in 96-well MicroAmp reaction plates(Applied Biosystems). Quantitative PCR reactions consisted of an initialdenaturation step of 95° C. for 10 min followed by 40 cycles of 95° C.for 15 s and 60° C. for 60 s. Each 96-well plate containing D. citrisamples included a no template control, a positive control (Las DNA inDNA extractions from D. citri), and a negative control (no Las DNA inDNA extractions from D. citri). Likewise, plates containing plantsamples included a no template control, a positive control (Las DNA inDNA extractions from plant), and a negative control (no Las DNA in DNAextractions from plant). Reactions were considered positive for eithertarget sequence if the cycle quantification (Cq) value, determined bythe ABI 7500 Real-Time software (version 1.4, Applied Biosystems), was≦32 [17].

Behavioral Bioassays

A custom designed two-port divided T-olfactometer [Analytical ResearchSystems (ARS), Inc. Gainesville, Fla.] that has been thoroughlydescribed in [98] was used to evaluate behavioral response of D. citri.Briefly, the olfactometer consisted of a 30 cm long glass tube with 3.5cm internal diameter that is bifurcated into two equal halves with aTeflon strip forming a T-maze. Each half served as an arm of theolfactometer enabling the D. citri to make a choice between twopotential odor fields. The olfactometer arms were connected to odorsources placed in 35 cm tall×15 cm wide dome shaped guillotine volatilecollection chambers (GVCC) (ARS, Gainesville, Fla.) through Teflon-glasstube connectors. The odor sources comprised 14-16 week old Las-infectedor non-infected C. sinensis plants placed in GVCC. Purified andhumidified air was pushed through the GVCC via two pumps connected to anair delivery system (ARS, Gainesville, Fla.). A constant airflow of 0.1L/min was maintained through both arms of the olfactometer. Theolfactometer was housed within a temperature-controlled room andpositioned vertically under a fluorescent 900-lux light bulb within afiberboard box for uniform light diffusion. This positioning tookadvantage of the negative geotactic and positive phototactic response ofD. citri [98].

D. citri adults were released individually into the inlet adapter at thebase of the olfactometer. An odor source was randomly assigned to one ofthe arms of the olfactometer at the beginning of each bioassay and wasreversed after every 30 insects to eliminate positional bias. Initially,D. citri adults were exposed to clean air vs. clean air in theolfactometer to verify the absence of positional bias. Thereafter, thebehavioral response of non-infected or Las-infected D. citri was testedagainst non-infected versus Las-infected plants. D. citri adults werealso exposed to non-infected grafted (sham grafted) vs.non-grafted-non-infected plants to determine the effects of graftingonly on D. citri behavior. For each treatment, C. sinensis plants weregrafted with C. aurantifolia budwood four months prior to initiatingbehavioral experiments. A minimum of 150 male or female D. citri adultswere examined per treatment combination (five replications of 30 D.citri). Each D. citri that responded to odors was individually subjectedto qPCR to determine Las infection status using the procedures describedabove.

Host Plant Selection Under Light or Dark Conditions

Light conditions. To evaluate settling preference of D. citri onnon-infected versus Las-infected plants, two non-infected and twoLas-infected plants of the similar age (14-16 week old) and vigor wereplaced randomly into a 0.35×0.35×0.6 m observation cage (BioquipProducts, Rancho Dominguez, USA). Thereafter, 240 D. citri adults (60per plant) were released into the center of each cage. The cages werehoused under temperature-controlled conditions of 27±1° C., 63±2% RH,and under a L14:D10 h photoperiod. There were four cages with each cagerepresenting a single replicate. The total number of D. citri settlingon each Las-infected or non-infected citrus plant was recorded three andseven days after release. Experiments were conducted separately and inan identical manner for either non-infected or Las-infected D. citri.Each D. citri adult settling on a non-infected or Las-infected citrusplant was individually tested using qPCR to confirm Las infection statusfollowing the procedures described above. Citrus plants used inexperiments were examined for Las infection four months after thecompletion of experiments.

Dark conditions. D. citri use a combination of olfactory and visual cuesto locate citrus host plants [37]. Visual cues such as light and colorof leaves may impact the settling preference of D. citri fornon-infected versus Las-infected citrus plants. Therefore, an experimentwas conducted under complete darkness to eliminate the role of visualcues. The materials and procedures were otherwise identical to thosedescribed above under light conditions. D. citri adults were countedthree and seven days after release using a source of red light.Preliminary testing indicated that red light did not affect psyllidbehavior. Furthermore, hemipterans lack red photoreceptors [99].

Movement of D. citri between Las-Infected and Non-Infected Plants

Given that initial orientation of insect vectors to plants could bedifferent from their final settling choices [55] and both could affectpathogen spread, we evaluated movement of D. citri between theLas-infected and non-infected plants. A non-infected or Las-infectedplant was inserted at random into an above described observation cage.Thereafter, 60 D. citri adults were confined on this plant using a meshsleeve for forced settling. Once all psyllids settled on the plant, anadditional, randomly selected non-infected or Las-infected plant wasinserted into the cage and placed 15 cm away from the initial plant.Immediately thereafter, the mesh sleeve was removed from the initialplant to allow psyllid movement between the two plants. The number of D.citri moving from the initial plant on which they settled and onto theinserted plant was counted seven days after release. Four combinationsof plant pairs were examined using either non-infected or Las-infectedD. citri for a total of eight treatments. The plant combinations testedwere: 1) Las-infected settling plant with a non-infected plantintroduction, 2) non-infected settling plant with a Las-infected plantintroduction, 3) non-infected settling plant with a non-infected plantintroduction, and 4) Las-infected settling plant with a Las-infectedplant introduction. The cages were housed in a temperature-controlledroom at 27±1° C., 63±2% RH, and under a L14:D10 h photoperiod. Eachcombination was replicated four times. Non-infected D. citri adultsfound moving from infected to non-infected plants were individuallyanalyzed for Las infection by qPCR as described above. Thereafter, theinserted non-infected plants were subsequently tested for nutrientstatus and possible Las infection due to movement of infected psyllids.Only female psyllids were used in these experiments because nodifferences were observed in behavioral responses between the sexes inpreliminary tests.

Feeding Efficiency of D. citri on Non-Infected Versus Infected Plants

One objective of these experiments was to compare feeding efficiency ofD. citri adults on Las-infected versus non-infected plants byquantifying their honeydew excretion. Four sets of experiments wereconducted to quantify honeydew excretion of psyllids after 24, 48, 72 or96 hr of feeding. Each experiment was replicated five times. Citrus leafdiscs obtained from Las-infected or non-infected plants were placedindividually on 1.5% agar beds within 60 mm plastic disposable Petridishes. Thereafter, ten D. citri adults of mixed age and sex (˜1:1) werereleased into each dish. Each Petri dish was sealed with lids lined with60 mm Whatman filter paper (Whatman International Ltd, Kent, UK). Petridishes were inverted to collect honeydew droplets onto the filter paper.Dishes were maintained at 25±1° C., 50±5% RH, and L14:D10 h photoperiodin an incubator. Collected filter papers were subjected to a ninhydrin(Sigma-Aldrich, St Louis, Mo.) test to facilitate counting of honeydewdroplets [100-101]. This technique, developed by Auclair [102], has beensuccessfully used to quantify honeydew excretion by psyllids,whiteflies, aphids and plant hoppers [100-101, 103-104].

Collection and Analysis of Plant Volatiles from Las-Infected orNon-Infected citrus Plants

Infected or non-infected plants, as described above, were confinedwithin a GVCC (ARS, Gainesville, Fla.). Charcoal-purified and humidifiedair was drawn over plants and pulled out at a rate of 300 mL min-1through a trap containing 50 mg of Super-Q adsorbent, 800-1000 mesh(Alltech Assoc., Deerfield, Ill., USA) for 24 hrs. Thereafter, Super-Qtraps were rinsed with 150 μl of dichloromethane into individual 2.0 mlclear glass vials (Varian, Palo Alto, Calif., USA, part number:392611549 equipped with 500 μl glass inserts). Volatiles were sampledfrom two plants simultaneously (one Las-infected and the secondnon-infected). A total of five plants that were previously identified asinfected and five similar plants identified as non-infected weresampled.

Collection of Volatiles from citrus Plants Before and During psyllidFeeding

Volatiles were collected to determine whether release of MeSA wasinduced by psyllid feeding. Plants were placed within a GVCC withidentical flow rates and collection procedures as described above.Volatiles were collected simultaneously from three undamaged plants (nopsyllid feeding) for 24 hrs, after which the adsorbent traps were rinsedinto vials for gas chromatography-mass spectrometry (GC-MS) analysis.Psyllids were divided by sex and randomly introduced into two of thethree plant chambers. Each infested plant received 300 psyllids (oneplant receiving only males and another plant receiving only females).The third plant was left uninfested as a control. Volatiles werecollected from plants for 24 hr, after which traps were rinsed intovials for GC-MS analysis. Also, volatiles were collected in this manneron three separate dates with six plants evaluated before and after D.citri introduction and three plants serving as simultaneous undamagedcontrols.

Analysis of Volatiles by GC-MS

An internal standard of n-octane (300 ng) was added to each sample priorto GC-MS analysis. A one μl aliquot of each extract was injected onto agas chromatograph (HP 6890) equipped with 30 m×0.25-mm-ID, 0.25 μm filmthickness DB-5 capillary column (Quadrex, New Haven, Conn., USA),interfaced to a 5973 Mass Selective Detector (Agilent, Palo Alto,Calif., USA), in both electron impact (EI) and chemical ionization (CI)modes. Helium was used as the carrier gas in the constant flow mode of30 cm/sec. The injector was maintained at 260° C. The oven wasprogrammed from 40 to 260° C. at 7° C./min. Isobutane was used as thereagent gas for CI, and the ion source temperature was set at 250° C. inCI and 220° C. in EI. The mass spectra were matched with NIST 2005version 2.0 standard spectra (NIST, Gaithersburg, Md.). The compoundswith spectral fit values equal to or greater than 90 and appropriate LRIvalues were considered positive identifications. When available, massspectra and retention times were compared to those of authenticstandards. Compounds were quantified as equivalents of the total amountof n-octane within each analyzed volatile collection sample. A moreaccurate quantification of MeSA was made by comparing the total ionchromatograph (TIC) EI response for standard solutions with knownquantities of n-octane and MeSA. A response factor of 1.2 was thenestablished for for MeSA relative to the n-octane based values.

Nutritional Status of Las-Infected and Non-Infected citrus Plants

The nutritional status of Las-infected and non-infected citrus plantswas analyzed by a commercial laboratory (Waters Agricultural Lab, Inc,Georgia). Leaf samples from 16 qPCR-confirmed Las-infected ornon-infected plants were washed, dried and ground to pass a 0.38-mmsieve. An individual leaf sample comprised about three g of dry leafbiomass obtained from three-month-old leaves. Leaf phosphorus,potassium, calcium, magnesium, sulfur, manganese, iron, copper, zinc,boron, silicon, sodium, molybdenum, nitrate, aluminum, and chlorineconcentrations were determined by inductively coupled plasma atomicemission spectroscopy.

Behavioral Response of D. citri to Synthetic Chemicals

MeSA was released in greater quantities from Las-infected plants thannon-infected plants, while MeAN and D-limonene were released in greaterquantities by non-infected than infected plants (See results).Therefore, the behavioral response of D. citri to these syntheticchemicals was tested. A known D. citri attractant, β-ocimene [38], wasused as a positive control. All chemicals were obtained from SigmaAldrich (St Louis, Mo.) with purities ranging between 97 and 99%. Purityof β-ocimene was 90% and contained a mixture of isomers comprising20-25% limonene. Each treatment was dissolved in 100 μl ofdichloromethane and pipetted onto a 2 cm Richmond cotton wick (PettyJohn Packaging, Inc. Concord, N.C.) at 0.001, 0.01, 0.1, 1.0, 10.0 and100.0 μg dosages. The control treatment consisted of a cotton wickimpregnated with solvent only. The solvent from both treatments wasallowed to evaporate within a fume hood for 30 min prior to assays. Thebioassay procedures with synthetic chemicals were identical to thosedescribed earlier for plant samples. However, in this case, treatments(odor sources) were placed into solid phase micro extraction chambers(SPMEC) (ARS, Gainesville, Fla.) instead of the GVCC described above.The SPMEC consists of a straight glass tube (17.5 cm long×2.5 cminternal diameter) supported with an inlet and outlet valve for incomingand outgoing air streams, respectively [100]. The treated and controlcotton wicks were wrapped in laboratory tissue (Kim wipes,Kimberly-Clark, Roswell, Ga.) to minimize contamination and placedrandomly into one of the two SPMEC enclosures that were connected to theolfactometer and the air delivery system through Teflon-glass tubeconnectors.

Only non-infected female psyllids were assayed in these experiments,because no differences in behavioral responses were observed between thesexes or between infected and non-infected psyllids in preliminarytests. At least 120 non-infected female adults were examined pertreatment combination. The treatment combinations evaluated in this setof experiment were 1) MeAN vs. clean air, 2) MeSA vs. clean air, 3)D-limonene vs. clean air, and 4) β-ocimene vs. clean air.

Data Analyses

Chi square (x²) tests were used to compare between the numbers of D.citri entering the treatment or control arm in the T-maze olfactometer.Numbers of D. citri settling on Las-infected versus non-infected citrusplants under light and dark conditions were analyzed using arepeated-measure, mixed-model, factorial analysis of variance (ANOVA)(Proc Mixed, Version 9.1, SAS Institute, Cary, N.C., USA). Las infectionstatus of citrus plants, Las infection status of D. citri, and gender ofD. citri were included as fixed effects. Means were compared usingTukey's Honestly Significant Difference (HSD) test. Significantdifferences in the number of honeydew droplets excreted on Las-infectedversus non-infected leaves were analyzed using a two-way ANOVA with Lasinfection status of leaves and D. citri exposure period as independentfactors. Means were compared using Tukey's HSD tests. In assays todetermine the movement of D. citri adults between Las-infected andnon-infected plants, the number of psyllids moving between the plantswas analyzed using one-way ANOVA, followed by means separation usingTukey's HSD test. D. citri adults that did not make a choice wereexcluded from statistical analyses. The nutrient data obtained fromLas-infected versus non-infected plants were analyzed using pairedt-tests. In all cases, the significance level was p<0.05.

The resulting volatile profiles were standardized as equivalents ofn-octane within each sample analyzed. The characteristic set ofvariables that defined a particular group (e.g. non-infected versusinfected plant) was found using the ‘varSelRFBoot’ function of thepackage ‘varSelRF’ for the ‘randomForest’ analysis (R software version2.9.0, R Development Core Team 2009). The varSelRF algorithm was usedwith Random Forests to select the minimum set of VOCs that werecharacteristic of differences between infected and non-infected plants.The tree-based Random Forests algorithm performs hierarchical clusteringvia multi-scale and combinatorial bootstrap resampling and is mostappropriate for data where the variables (VOCs in this case) outnumberthe samples, and where the variables are auto correlated, which is atypical problem of conventional multivariate analysis of such data.Consequently, this type of analysis is common in bioinformatics,chemoinformatics and similar data-rich fields. Two hundred bootstrappingiterations of the Random Forests algorithm were employed to arrive at aminimal set of VOCs that could differentiate between infected andnon-infected plants. The mean decrease in accuracy (MDA) was calculatedwhen individual VOCs are removed from the analysis. MDA values indicatethe importance value of particular VOCs for the discrimination betweentreatments. In addition, treatment differences between individualvolatiles collected from headspace of infected and non-infected plantswere compared using paired t-tests.

Results

Response of psyllids to Host Plant Odors

The majority (>80%) of those D. citri tested responded to the odors ofeither non-infected or Las-infected citrus plants. Significantly more D.citri males (χ²=4.32, df=1, P=0.04) and females (χ²=6.53, df=1, P=0.01)were attracted to the odors from Las-infected plants than non-infectedplants (FIG. 1). Las-infected male (χ²=5.24, df=1, P=0.02) and female(χ²=4.37, df=1, P=0.04) psyllids were also more attracted to the odorsfrom infected than non-infected plants (FIG. 1). No significantdifferences were detected when D. citri adults were exposed tonon-infected grafted (sham grafted) vs. non-grafted-non-infected plants.

Host Plant Selection Under Light or Dark Conditions

Light conditions. Observation time, Las infection status of D. citri,and gender of D. citri did not significantly affect the number of D.citri that settled on Las-infected versus non-infected plants. Lasinfection status of plants significantly interacted with observationtime (F=160.07, df=1.30, P<0.001). More adult D. citri (of either genderand infection status) landed upon Las-infected plants than onnon-infected plants three d after release (FIG. 2A). However, more adultD. citri were found on non-infected plants than on Las-infected plantsseven d after release (FIG. 2A). Non-infected plants remainedLas-negative when examined four months after exposure to Las-infected ornon-infected D. citri in behavioral assays.

Dark conditions. Observation time, Las infection status of D. citri, andgender of D. citri did not significantly affect the number of D. citrithat settled on Las-infected versus non-infected plants. Las infectionstatus of plants significantly interacted with observation time(F=49.45, df=1.30, P<0.001). More adult D. citri (of either gender andinfection status) landed on Las-infected plants than on non-infectedplants three d after release (FIG. 2B). However, more adult D. citriwere found on non-infected plants than on Las-infected plants seven dafter release (FIG. 2B). Non-infected plants remained Las-negative whenexamined four months after exposure to Las-infected or non-infected D.citri in behavioral assays.

Movement of D. citri Between Las-Infected and Uninfected Plants

Movement of non-infected or Las-infected D. citri from Las-infected tonon-infected plants was greater than observed for any other treatmentcombination (F=7.69, df=7.24, P<0.0001) (FIG. 3A, B). More Las-infectedD. citri tended to move than uninfected counterparts after initialsettling, although this difference was not statistically significant.Approximately 5% of non-infected psyllids moving from infected tonon-infected plants acquired the Las bacterium (Table 1). Four monthsafter the termination of the experiment, all of the non-infected plantsthat had been inserted into cages with Las-positive plants testedpositive for Las (Table 1). Similarly, all of the non-infected plantsinserted into cages with Las-positive plants tested positive for Laswhen known Las-infected psyllids were used (Table 1).

TABLE 1 Las infection status of inserted plants and Diaphorina citrimigrating from initial point of forced settling to subsequently insertedplant treatments. % of % of inserted Las-infected Las infection plantsD. citri on Inserted status of infected inserted Initial plant plantreleased D. citri with Las plants Non-infected Infected Non-infected 1000.0 Non-infected Infected Infected 100 71.2 Non-infected Non-infectedNon-infected 0.0 0.0 Non-infected Non-infected Infected 100 68.9Infected Infected Non-infected 100 6.6 Infected Infected Infected 10076.3 Infected Non-infected Non-infected 0.0 4.8 Infected Non-infectedInfected 100 69.7Feeding Efficiency of D. citri on Non-Infected Versus Infected Plants

The number of honey dew droplets produced by psyllid feeding, asurrogate measure of feeding efficiency, was significantly affected bythe infection status of plants (F=65.99, df=1.39, P<0.0001), feedingexposure time (F=81.81, df=3.24, P<0.0001), and the interaction betweenthe two factors (F=7.11, df=3.28, P=0.0011). There was no significantdifference in the amount of feeding on non-infected versus infectedplants after 24 h of feeding by psyllids (FIG. 4). However, psyllids fedsignificantly more on non-infected than on Las-infected plants after 48,72 and 96 h, respectively.

Volatile Release by Las-Infected and Non-Infected citrus Plants

There were significant qualitative and quantitative (Table 2, FIG. 5)differences between the headspace volatiles of non-infected andLas-infected plants (Table 2). Las-infected plants releasedsignificantly more MeSA than non-infected plants (Table 2, FIG. 5),while non-infected plants released more methyl anthranilate (MeAN) andD-limonene than infected plants (Table 2). Using a Random Forestsalgorithm, a minimum of two compounds, MeAN and MeSA, were identifiedthat discriminated between the infected and non-infected volatileorganic compound (VOC) signatures of these treatments, with an estimateprediction error of 0.15 and a ‘leave-one-out’ bootstrap error of 0.20.

TABLE 2 Volatiles from Las-infected and non-infected citrus plantspresented as average percentage (±1 SE) of n-octane equivalents ofvolatile organic compounds collected from plants' headspace. Eachcompound is characterized by its retention time (RT) and major ion. Las-P- Compound RT CAS#* Uninfected ±SE infected ±SE value Sabiene 7.943387-41-5 11%  2.54 15%  0.67 0.68 β-pinene 8.01127-91-3 >0.01%    >0.001 >0.01%    >0.001 0.89 Myrcene 8.20 123-35-3 3%0.44 3% 0.23 0.84 3-Carene 8.57 13466-78-9 5% 1.09 2% 0.08 0.35D-limonene 8.71 5989-27-5 59%  3.18 27%  0.79 0.02* β-ocimene 8.95502-99-8 6% 1.01 6% 0.50 0.82 Linalool 9.83 78-70-6 2% 0.56 1% 0.11 0.72Menthatriene 10.14 18368- >0.01%    >0.001 4% 0.25 0.13 (1,3,8-para)95-1 Methyl 11.01 119-36-8 1% 0.21 39%  2.86 0.05* salicylate Geranial12.21 141-27-5 2% 0.45 0% 0.0 0.15 Methyl 13.90 134-20-3 12%  1.25>0.01%    0.05 0.04* anthranilate Caryophyllene 14.45 87-44-5 >0.01%   0.02 3% 0.22 0.16 Bold values indicate a significant difference betweentreatments (P < 0.05). The chemicals which were present in differentproportions between Las-infected and non-infected citrus plants areshown. Identification was based on comparisons of retention times withstandard and spectral data from Adams, EPA, Nist05 Libraries andsynthetic standard comparison. *CAS: Chemical Abstract ServiceVolatile Release Before and During psyllid Feeding

MeSA was only detected in headspace volatiles from plants after psyllidswere introduced and allowed to feed. It was detected in headspaceprofiles from plants infested with females as well as males (Table 3).Analysis of the volatile profiles of plants prior to the introduction ofpsyllids of either sex or from negative control plants yielded no MeSA(Table 3).

TABLE 3 Detection of methyl salicylate release from plants prior to andduring Diaphorina citri feeding. Presence of methyl salicylate TreatmentDay 1* Day 2 Female feeding − + Male feeding − + Undamaged control − −*Plants sampled on Day 1 were not exposed to psyillds. Samples on Day 2received 24 h of psyllid feeing. − indicates no detection while +indicates positive detection of the volatile.

The amount of MeSA released (mean±SE) by plants exposed to psyllidfeeding (10.3±3.00 ng/plant/24 hr) was similar to the amount released byLas-infected plants (12.87±4.78 ng/plant/24 hr), and both of theseamounts were similar to the quantity of synthetic MeSA found to beattractive to psyllids in behavioral bioassays (Table 4).

TABLE 4 Responses of Diaphorina citri when assayed with syntheticvolatiles identified from Las-infected and non-infected citrus plants.Proportion Proportion of D. citri of D. citri responding to Dosageresponding to control χ² P Chemical (μg) treatment arm arm value value†β-ocimene 0.001 52.22 47.78 0.18 0.67 (positive 0.01 54.46 45.54 0.80.37 control) 0.1 53.19 46.81 0.38 0.54 1.0 60.44 39.56 3.97 0.05* 10.061.46 38.54 5.04 0.02* 100.0 62.77 37.23 6.13 0.01* D-limonene 0.00156.06 43.94 0.97 0.32 0.01 60.87 39.13 2.17 0.14 0.1 54.39 45.61 0.440.51 1.0 54.65 45.35 0.74 0.39 10.0 60.24 39.76 3.48 0.06 100.0 61.5438.46 4.85 0.03* Methyl 0.001 54.00 46.00 0.32 0.57 salicylate 0.0160.44 39.56 3.97 0.04* 0.1 54.76 45.24 0.38 0.54 1.0 45.12 54.88 0.780.38 10.0 41.03 58.97 2.51 0.11 100.0 39.56 60.44 3.97 0.04* Methyl0.001 58.06 41.94 0.81 0.37 anthranilate 0.01 57.14 42.86 0.71 0.40 0.161.76 38.24 1.88 0.17 1.0 57.33 42.67 1.61 0.20 10.0 56.10 43.90 1.220.27 100.0 52.27 47.73 0.23 0.63 †β-ocimene carried 20-25% limonene. Pvalues labeled with * are significantly different (χ² test, P ≦ 0.05).Rows highlighted in green indicate attraction while rows highlighted inorange indicate repulsion.Behavioral Response of D. citri to Synthetic Chemicals

Behavioral bioassays with synthetic chemicals identified fromLas-infected and non-infected plants revealed that D. citri wereattracted to MeSA at the 0.001 μg dosage (χ²=4.85, df=1, p=0.03), butthat they were repelled by this chemical at the 100 μg dosage (χ²=4.44,df=1, p=0.04) (Table 4). D. citri were attracted to D-limonene (χ²=4.85,df=1, p=0.03) only at the 100 μg dosage, while MeAN did not attract orrepel D. citri at any of the dosages tested. The positive control(β-ocimene) was attractive at 1-100 μg dosages (Table 4).

Nutritional Status of Las-Infected and Non-Infected citrus Plants

There were marked differences between infected and non-infected plantswith respect to nutrient content (Table 5). Las-infected plants weredeficient in N, P, Mg, Zn, and Fe as compared with non-infected plants(Table 5). However, Las-infected plants had higher K and B contentscompared with non-infected plants (Table 5). There were no differencesbetween Las-infected and non-infected plants for Ca, S, Si, Mn, Na, Mo,Al, and Cl.

TABLE 5 Differing levels of various nutrients between Las-infected andnon-infected Citrus sinensis plants. Plant status N P K Mg Ca S B Zn MnFe Si Na Mo Al Uninfected plant 3.13 0.17 3.00 0.43 1.88 0.25 46.6729.33 39.00 86.33 0.15 0.08 0.02 35.34 Las-infected plant 2.32 0.11 4.050.29 1.75 0.24 76.00 19.67 35.33 66.67 0.16 0.11 0.01 16.98 T value 8.033.72 19.10 8.67 1.67 0.42 12.35 45.85 1.21 16.75 0.43 1.69 0.42 2.08 Pvalue <0.01* 0.01 <0.01* <0.01* 0.15 0.69 <0.01* <0.01* 0.28 <0.01* 0.680.14 0.69 0.09 Nutrient values in columns labeled with * aresignificantly different at p < 0.05 (paired t-Test). Values for N, P, K,Mg, Ca, S, Si, and Na are in % while values for other nutrients are inppm.

Discussion

The above results indicate that Las-infected plants were initially moreattractive to D. citri adults than non-infected plants; however,psyllids dispersed subsequently to non-infected plants after initiallysettling on infected plants. Similar results obtained with thebehavioral responses of D. citri under both light and dark conditionssuggest that initial movement of psyllids to Las-infected plants islikely mediated by volatile cues. Infection of citrus with this plantpathogen induced release of MeSA, which attracted D. citri. A similaramount of MeSA was also released by the trees in response to D. citriinfestation, suggesting that the same cue exploited by the pathogen toattract its vector may be used by the vector to locate congregations ofconspecifics feeding on non-infected plants. Following initialchemically mediated attraction to infected plants, psyllids tended tosubsequently disperse to non-infected plants, making them theirpreferred settling choice. While not wishing to be bound by theory, thismay have been due to the sub-optimal quality of infected plants ascompared with non-infected plants, as evidenced by lower feedingefficiency on infected than non-infected leaves. Importantly, thispathogen-manipulated behavioral sequence facilitated pathogen spread bythe vector.

The above results further suggest that an insect-transmitted bacterialplant pathogen alters plant traits so as to induce odor-mediatedbehavior from the vector that may ultimately benefit the pathogen. Whileincreased preference of insect vectors to virus-infected versusnon-infected host plants has been demonstrated for both persistently andnon-persistently transmitted viruses [3,4,9], little is known aboutsimilar interactions involving pathogenic bacteria. Furthermore, priorstudies on pathogen-vector interactions have focused on singlemechanisms (olfactory, visual or nutritional) [13-14,59-62]. In thecurrent investigation, the inventors sought to determine severalunderlying mechanisms (chemical, visual, and nutritional) that affectthe behavior of an insect (D. citri) in response to plant infection bythe pathogen (Candidatus Liberibacter asiaticus) that it transmits.

Volatiles from infected and non-infected citrus are attractive to D.citri as confirmed by the current data and previous studies [38]. D.citri were attracted to common volatiles released by citrus such asβ-ocimene and D-limonene, implicating these as general host selectioncues. Both β-ocimene and D-limonene are predominant citrus volatilesreleased by citrus flush, foliage, and fruit [38,63-65]. However,pathogen infection induced release of certain novel components or aquantitative increase in release of certain compounds that were releasedin lower quantities by non-infected plants. Among the novel chemicalsidentified that distinguished Las-infected from non-infected plants,only MeSA elicited attraction from D. citri and may explain the enhancedattractiveness of infected plants. Our results, showing induced releaseof novel volatiles from infected plants, contrast with previous resultsdocumenting changes in the ratio of volatiles released after infectionby persistently transmitted viruses [3,4,] or an increase in the releaseof existing volatiles (no change in blend composition) followinginfection by non-persistent plant viruses [9]. In the current example,bacterial infection of plants induced release of a novel compound(MeSA). MeSA alone elicited attraction from D. citri at a similarconcentration at which it was released from Las-infected plants.

Movement of psyllids from infected to non-infected plants after initialselection of infected plants suggests that their initial response toolfactory cues may not be directly linked to the most beneficial hostplant. This was also confirmed by feeding assays measured by honeydewexcretion. These results suggest that psyllids must feed on infectedplants in order to discern poor quality of the host. Therefore,volatiles appear to be involved in host finding, but not in arrestmenton the host. Feeding is required for insects to differentiate betweennon-infected and infected plants and gustatory cues are involved in hostacceptance [55,68-69].

Volatile collections from psyllid-infested plants detected inducedrelease of MeSA, suggesting a coincidental convergence on a single cuethat may simultaneously benefit the pathogen by deceptively attractingits vector, which also uses this cue to locate conspecifics or identifyvulnerable hosts. These results suggest a potential deceptive trade-offfor the herbivore. Although plants infested by conspecifics may exhibitcompromised defenses, those infected by the pathogen appear to be lesssuitable than non-infected hosts; however, both phenotypes releasenearly the same amount of the MeSA attractant cue. This may explain whyour results are not congruent with the recent hypothesis proposed byMauck et al. [9] that a “deceptive host phenotype” (more attractive, butless suitable) should attract the vector by increasing overall releaseof attractive volatiles quantitatively instead of producing novel (notreleased by non-infected plants) cues.

Pathogen-induced release of plant volatiles has been previouslyestablished following virus infection. In these cases, CMV, PLRV, andBYDV induced release of volatiles that rendered infected plants moreattractive to their aphid vectors than non-infected plants. Both PLRVand BYDV infections improved the quality of the host plants to theirrespective vectors [3,4,76-77], resulting in preferential arrestment onthe infected plants due to contact and gustatory cues [3,4,56] Thepresent results are similar to the findings of Mauck et al [9], wheresquash plants infected with CMV released volatiles that initiallyattracted the aphid vector, but were poor hosts for their vectors,causing subsequent movement from infected to non-infected plants.Pathogen-mediated manipulation of vector behavior may depend on howpathogens are transmitted. Persistent pathogens, which require longerfeeding durations for acquisition, benefit from increased arrestment oftheir vector, while non-persistent or semi-persistent pathogens mayinduce changes in plant phenotype that cause vectors to quickly dispersefollowing acquisition [9]. In the above experiments, psyllids wereinitially attracted to infected plants, but later preferred to settle onnon-infected hosts. Although D. citri were initially attracted toinfected plants, which was likely mediated by a volatile cue (consistentwith both persistent and non-persistent virus examples [3,9], psyllidspreferentially dispersed to non-infected plants after initial feeding oninfected ones (consistent with non-persistent virus example [9].

Pathogen spread may be favored in an environment with a low frequency ofinfected plants, when vectors prefer infected plants [78]. However, inan environment with a high frequency of infected plants, pathogen spreadmay be favored when vectors prefer non-infected plants [78]. More recentmathematical modeling suggests that when vectors prefer to orient toinfected plants, pathogen spread is slow when the frequency of plantinfection is low, but pathogen spread is rapid when most plants areinfected [55]. Moreover, vector preference for infected versusnon-infected plants is partitioned into orientation preference andfeeding preference and these two distinct suites of behavior affectpathogen spread differently, depending on whether vectors preferinfected versus non-infected plants [55]. While feeding preference forinfected plants may decrease pathogen spread, orientation preference forinfected plants may lead to rapid pathogen spread [55]. Acquisition ofLas by D. citri can occur after 30 minutes to 24 hr of feeding [28,31].Therefore, initial landing by psyllids on infected plants for 24 hr orlonger is sufficient for acquisition of the pathogen.

Subsequent movement of psyllids from infected to non-infected plantswill result in inoculation of new plants as demonstrated by the currentresults. Therefore, manipulation of D. citri behavior due to Lasinfection of plants may increase pathogen spread in the field; however,this will likely depend on the frequency of plant infection [55]. Theeffect of vector preference on pathogen spread depends on several otherfactors, including the mode of pathogen transmission, latency period,vector movement behavior, and the combined dynamic spatial patterns ofthe plant, pathogen, and vector [55,78]. Given the apparent convergenceon the same attractant cue released in response to Las infection andvector feeding, it is difficult to speculate under what conditionspathogen spread would be favored in the field. Attraction of D. citri toMeSA suggests a possible conflict of interests between the pathogen andthe vector. In the early phase of the infection, more conspecifics wouldbe expected to congregate on non-infected plants than on infectedplants. Thus, the frequency of non-infected plants releasing MeSA wouldbe greater than the frequency of Las-infected plants releasing thisattractant, resulting in greater attraction of psyllids to non-infectedplants. Therefore, preferential initial attraction to infected plantsmay occur either under low population densities of the vector and whenthe plant infection rate is low, and/or only when the plant infectionrate is high.

Furthermore, attraction of vectors to hosts of suboptimal qualitysuggests another “conflict of interest” [9] between the pathogen and itsvector, given that vector performance may be lower on plants, whichserve as reservoirs of the pathogen. However, in the D. citri-Lasinteraction, initial attraction to and subsequent dispersal frominfected plants could benefit both the pathogen and vector. Vectorfeeding on infected host plants promotes acquisition and spread of thepathogen, while infection with the pathogen may have positive effects onvector fitness. Las-infection appears to benefit the fitness of D. citriby increasing fecundity [Pelz-Stelinski, unpublished results].

Las-infected plants were deficient in N, P, Mg, ZN and Fe, but werecharacterized by higher concentrations of K and B. While not wishing tobe bound by theory, it is possible that infected plants are asub-optimal host for D. citri because of a nutritional imbalance causedby Las infection. Phloem-feeding plant hoppers (Prokelisia dolus)disperse to higher quality Spartina plants when reared on N and Pdeficient plants [79]. Also, increased N content improves theperformance of leaf-feeding aphids (Metopolophium dirhodum) on wheat andbarley [80-81]. Physiologically, N is essential for insect growth,survival, and reproduction due to its fundamental role in proteinsynthesis [82-86]. Phosphorus acts against viral disease by promotingplant maturity, thus restricting pathological effects of virus infection[87]. However, plant nutrient deficiencies can have a negative orpositive effect on population dynamics of phloem feeding insects. Forexample, K deficiency in soybean increases fecundity and populationgrowth of soybean aphid [88-90].

The nutrient analyses described in the current study only addressed theelemental composition of leaf tissues. There may be several otherunidentified factors including sugars, amino acids, and proteins thatcould have contributed to the movement of D. citri from infected tonon-infected plants. D. citri feeds specifically within phloem cells,obtaining nutrition from free amino acids, proteins and sugars [26],which are known to affect vector growth [2,69,91-92]. Furthercomparisons of the elemental composition of psyllids and phloem tissuesshould help elucidate how host quality affects movement of D. citri.Las-infected leaves have been reported to accumulate up to 7.9-fold morestarch than non-infected leaves, resulting in blockage of the phloemvessels [93]; however, the effect of starch accumulation on D. citrinutrition is still unknown. Starch accumulation and callose formation inrice leaves is reported to increase plant resistance to the brown planthopper, Nilaparvata lugens, by preventing phloem ingestion [94]. In asimilar experiment, plants infected with Zucchini yellow mosaic virus(ZYMV) were characterized by lower total protein and sugar content thannon-infected plants [69]. However, both ZYMV-infected and non-infectedplants had identical total amino acid contents and Aphis gossypii livedlonger and produced more offspring on infected than on non-infectedplants [69].

In conclusion, Las-infected plants were initially more attractive to D.citri adults than non-infected plants; however, psyllids dispersedsubsequently to non-infected plants to make them their preferredlocation of settling rather than infected plants. The duration ofinitial setting was sufficient for D. citri to acquire the Las pathogen.Thus, the pathogen may be modifying the behavior of the vector byinducing changes in the attractiveness of the host plant througholfactory cues. This scenario suggests a mechanism for spread of thepathogen in the field because initial attraction and feeding of D. citrion infected host plants should facilitate acquisition of the pathogen,while subsequent movement away from potentially sub-optimal infectedplants should facilitate inoculation of non-infected plants. Overall,this behavioral manipulation of the vector by the action of the pathogenon the plant favored spread of the pathogen in a laboratory setting. Thepresent results indicate that MeSA is a specific chemical cue mediatinginitial psyllid attraction to Las-infected plants.

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While various embodiments of the present invention have been shown anddescribed herein, it will be obvious that such embodiments are providedby way of example only. Numerous variations, changes and substitutionsmay be made without departing from the invention herein. Accordingly, itis intended that the invention be limited only by the spirit and scopeof the appended claims.

The invention claimed is:
 1. An insect attractant comprising: an amountof methyl salicylate effective to attract a plurality of vectors ofcitrus greening disease; and an agriculturally acceptable carrier. 2.The insect attractant of claim 1, wherein the vectors comprise at leastof D. citri and Triosa erytreae.
 3. The insect attractant of claim 1,wherein the vectors are a carrier of the pathogen CandidatusLibreribacter asiaticus.
 4. The insect attractant of claim 1, whereinthe amount of methyl salicylate in the attractant is from 0.001 μg to200 μg.
 5. The insect attractant of claim 1, further comprising acompound selected from the group consisting of β-ocimene, D-limonene,methyl anthranilate, and combinations thereof.
 6. (canceled)
 7. A methodfor inhibiting infestation of a citrus plant by vectors of citrusgreening disease, the method comprising: identifying a target area ofdesired citrus plant growth; and applying an amount of an insectattractant comprising methyl salicylate to an area outside of the targetarea effective to draw the vectors of citrus greening disease away fromthe target area.
 8. The method of claim 7, wherein the area outside ofthe target area is at least 0.0001 m, 0.1 m, 0.5 m, 1 m, 2 m, 10 m, 50m, 100 m, or 1000 m from a point on a perimeter of the target area. 9.The method of claim 7, wherein the vectors comprise at least of D. citriand Triosa erytreae.
 10. The method of claim 7, wherein the vectors area carrier of the pathogen Candidatus Libreribacter asiaticus.
 11. Themethod of claim 7, wherein the amount of methyl salicylate in theattractant is from 0.001 μg to 200 μg.
 12. The method of claim 7,further comprising a compound selected from the group consisting ofβ-ocimene, D-limonene, methyl anthranilate, and combinations thereof.13. (canceled)
 14. A method of ameliorating spread of citrus plantpathogen amongst a plurality of trees, said method comprising applyingan effective amount of methyl salicylate to the trees, whereby theapplication of methyl salicylate masks signals from an infected tree.15. The method of claim 14, wherein said plurality of trees is an orangegrove.
 16. The method of claim 14, wherein said effective amount ofmethyl salicylate comprises an amount sufficient to diminish an abilityof an insect vector to discern between an infected tree and annon-infected tree.
 17. The method of claim 14, wherein the citrus plantpathogen is one that causes citrus greening.
 18. The method of claim 14,wherein said effective amount is at a concentration and amountsufficient to attract insect vectors.
 19. The method of claim 14,wherein said effective amount is at a concentration and amountsufficient to repel insect vectors.
 20. The method of claim 16, whereinthe insect vectors comprise at least of D. citri and Triosa erytreae.21. (canceled)